Project Summary/Abstract The voltage gated proton channel (HV1) exists in many human tissues and plays numerous roles vital to human health. For example, it contributes to bacterial killing by white blood cells, sperm maturation and mobility, histamine release by basophils, B lymphocyte signaling, and airway fluid regulation. Abnormal HV1 function has been implicated in breast cancer metastasis, brain damage in ischemic stroke, and exacerbation of chronic lymphocytic leukemia. As its gene was not reported until 2006, HV1 is a newcomer to the voltage gated ion channel family. Finally, its structure is unique in resembling a crucial component of all voltage-gated ion channels. This newcomer status, its unique structure, and its essential roles in human health and disease make understanding HV1 function and dysfunction highly significant. Directly translational studies will evaluate reported involvement of HV1 in breast cancer growth and metastasis. Tumor growth in mice will be examined using cells with different HV1 expression levels, ranging from complete knock-out (CRISPR/Cas9) to reduced (shRNA) to normal (WT). Our current working hypothesis is that HV1 acts as a switch that transduces membrane potential changes into cellular pathology. We will also build on our discovery of the involvement of HV1 in human B cells and in chronic lymphocytic leukemia. A novel approach will be to determine the effects of mutations indentified in human subjects with B cell malignancy. The DeCoursey lab has been deeply involved in the study of HV1, from discovering its existence in mammalian and human cells, to identifying its role in a number of human cells and tissues, to finally dissecting the molecule itself to identify which parts perform the major functions. Over the next five years we intend to pursue expanding our knowledge of this important molecule at multiple levels, building on our recent progress. We found that the mechanism producing proton selective conduction requires an aspartate in the center of the pore. We will test whether a hydrophobic region plays an additional critical role using mutagenesis, patch-clamp, and molecular dynamics simulations. We will attack the mechanisms of voltage-gating and the unique ?pH dependent gating that is essential to all functions of this molecule using similar approaches, but including a detailed mechanistic model as well as a newly improved molecular dynamics approach that determines protonation empirically rather than assuming it. We will continually refine our knowledge of the structures of both closed and open HV1 channels, using histidine scanning mutagenesis and NMR. Structure-function knowledge is crucial both for understanding mechanisms and for drug design.